Diarrheal antitoxin

ABSTRACT

This disclosure teaches a method of inactivating bacterial toxins, such as produced by Vibrio cholerae and Escherichia coli (hereinafter V. cholerae and E. coli) that are responsible for diarrhea in warm-blooded animals, comprising the placing of an effective dosage of ascorbic acid salts thereof, or derivatives such as the ester ascorbyl palmitate in the intestinal tract. 
     Definition: Ascorbic acid, salts thereof, and derivatives thereof such as ascorbyl palmetate have been identified as suitable detoxifying material according to this invention. For the sake brevity, the term &#34;ascorbic acid&#34; hereinafter shall include the salts and esters. 
     Diarrhea as used herein will include other toxins effecting the lower duodenum and intestines.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention described the innovative use of L-ascorbic acid as orallyapplied to the gastrointestinal tract. The L-ascorbic acid counteractsthe toxins known to contribute to diarrhea secondary to bacterialinfection.

2. Description of Prior Art

Numerous articles have been published in medical textbooks regardingbacterial diarrhea, its symptoms and its etiology (Scientific AmericanMedicine, Volume 7, No. 6).

The following background information, substantially as set forth inMerck Manual 13th Edition, Chapter 12, page 788, will assist inunderstanding the discovery of this invention:

Etiology

Gastroenteritis is a generic term, often implying a non-specific,uncertain, or unknown etology. However, certain diseases of knownbacterial, viral, parasitic, or toxic etology can also be included inthe clinical definition. Indeed, when a specific etiology can beidentified, the less specific term (gastroenteritis) can be avoided.Such bacterial dysenteries as cholerae, salmonellosis, and shigellosisshare common pathologic mechanisms and can be considered prototypes forsyndromes of lesser specificity.

Pathophysiology

Certain bacterial species elaborate exotoxins (enterotoxins) whichimpair intestinal absorption and can provoke secretion of electrolytesand water. In some instances, such as the enterotoxin of V. cholerae, achemically pure toxin has been characterized. Pure toxin alone willproduce a voluminous watery secretion from the small intestine seenclinically, thereby demonstrating an adequate pathogenic mechanism forthe diarrhea. Interotoxins probably explain other diarrhea syndromespreviously attributed to non-specific causes. For example, E. colitoxins has been determined to be the cause of some outbreaks of "nurserydiarrhea" and "traveler's diarrhea". Non-cholera vibrosis elaborates atoxin which appears to be responsible for some types of food poisoningdue to shellfish.

In addition to the above substantially quoted background, the literaturenotes that some shigella, salmonella and even E. coli species penetratethe mucosa of the small bowel or colon and produce microscopiculceration, bleeding, exudation of protein-rich fluid and secretion ofelectrolytes and water. This may occur whether or not the organismelaborates a toxin. This invention is directed to the prevention and/ortreatment of those types of diarrhea caused by toxins.

Traveler's diarrhea syndrome causes wide spread difficulty fortravelers, especially in underdeveloped parts of the world. Thepathogenic strain of E. coli is endemic to the parts of the world thatlack the hygienic facilities of the United States and countries ofWestern Europe. Specific therapy for preventing this syndrome has beenlacking, and only symptomatic anti-diarrheal therapy has been available.

SUMMARY OF THE INVENTION

This invention is the discovery that L-ascorbic acid, salts thereof, orderivatives such as the ester ascorbyl palmitate when applied to thegastrointestinal tract of humans and other warm-blooded animals, willinactivate toxins that contribute to traveler's diarrhea.

Although the toxin which is responsible for traveler's diarrhea isessentially that produced by certain strains of pathogenic E. coli.,there are other toxins produced by other bacteria which are essentiallyidentical from chemical and biological points of view (i.e. choleraetoxin produced by V. cholerae).

The novel discovery and approach of this invention is the focus ondetoxification of the toxic product of bacteria, at the location of thetoxin, rather than an attempt to eliminate the bacteria.

It has been discovered, according to this invention, that L-ascorbicacid is outstandingly effective in detoxification of the toxins found inthe instestinal tract of a human host. Although L-ascorbic acid is knownto be a strong antioxidant, it is not known how the L-ascorbic acidinactivates the toxin. Toxin structures are as yet unknown, and thechemistry of this invention is unknown.

It is known by the discovery of this invention that the externaladministration of an effective amount of ascorbic acid throughout thelumen of the small intestine and large intestine of a human host priorto and during exposure to toxin producing bacteria will detoxify anytoxins to the point of substantially complete elimination of the dangerof traveler's diarrhea and similar toxin induced distress.

GENERAL DISCLOSURE

This disclosure explains the discovery that the toxins which causediarrhea are inactivated by L-ascorbic acid and its salts. TheL-ascorbic acid is administered orally as a product which is intendedfor use in preventing and/or treating diarrhea. The invention alsodescribes the need for pharmaceutical forms of ascorbic acid that remainwithin the lumen of the gut and are not systematically absorbed. Inorder for ascorbic acid to be effective, it must inactivate thecausative toxins at the location of the toxin which is the lumen of thegut, especially the lower duodenum, ileum and jejunum. Free ascorbicacid administered orally in all available dose forms, either as freeascorbic acid, or its salts or derivative or in sustained release form,is inadequate as an antitoxin because it is rapidly absorbed from thestomach and upper duodenum.

This invention discloses a means to specifically inactivate thecausative bacterial toxins by a chemical reaction. The chemical reactionutilizes forms of L-ascorbic acid that are active in the smallintestine. This invention is useful both as a prophylactic andtherapeutic means to treat traveler's diarrhea and cholerae.

DESCRIPTION OF THE PREFERRED EMBODIMENT

L-ascorbic acid has important properties in that the dry crystals arestable in the air for a very long period of time. However, onceL-ascorbic acid enters into solution, it is capable of undergoingoxidation in a variety of reactions. The tendency of L-ascorbic acid tobe oxidized increases with increasing pH ("The antioxidant Vitamins,"CRC Critical Review, Food Sciences and Nutrition, March, 1979, pp. 271).

L-ascorbic acid is added, according to this invention, to an appropriatecarrier at milligram levels, which is completely compatible with the pHof the intestinal tract.

Although it has not been determined how L-ascorbic acid functions todetoxify bacterial toxins, it is known that L-ascorbic acid possessesrelatively strong reducing power as is shown in its ability todecolorize many dyes (Merck Index, 8th ed.). These kinds of reactionsmay be accelerated by alkalies, iron and copper.

There appears to be no known approach to traveler's diarrhea preventionusing L-ascrobic acid. This new use of a very safe product has beendiscovered, according to this invention, to possess essentially full andcomplete power for inactivation of toxins responsible for traveler'sdiarrhea.

EXAMPLE A

The mechanism by which L-ascorbic acid renders the E. coli and choleratoxins ineffective is unknown, but L-ascorbic acid will act as areducing agent, an antioxidant and a free radical sequestering agent.From in vitro testing of this invention it has been shown that ascorbicacid does inactivate the causative agents in traveler's diarrhea.L-ascorbic acid is known to oxidize to dehydroascorbate. Therefore, itis theorized that the detoxification may be the result of the followingreaction: ##STR1##

By a series of intermediate reaction steps, a protein or toxin mayinteract with the ascorbic acid and any intermediates to break thedisulphide bond and produce reduced sulfhydral groups. The reaction isrepresented as follows: ##STR2##

It is necessary to emphasize that this invention is a discovery, andthat the actual mechanism of detoxification is not yet known.

EXAMPLE B

The efficacy of the ascorbic acid in detoxifying bacterial toxins isdemonstrated in an animal model. The ascorbic acid is supplied via asurgically implanted catheter to the region of the intestine which issusceptible to toxin action. Two different animals (i.e. rabbits) areused, one for receiving toxin alone and the other toxin plus ascorbicacid. The dose of Cholera Toxin is 20 μg/kg and the dose of ascorbicacid is 10 mg/kg. Both agents are admininstrated in pH 6.5 phosphatebuffered saline. The control rabbit received only toxin and thetreatment rabbit received both toxin and ascorbic acid. The controlrabbit exhibited the classical diarrhea symptoms while the treatmentrabbit was unaffected by the toxin.

In the preceding disclosure, there has been no reference to catalyticagents, and, in fact, catalytic agents are believed to be unnecessary inmost instances. There are sufficient metallic ions present in mostenvironmental situations to serve any catalytic requirements of theoxidation of L-ascorbic acid. Nevertheless, in order to assurecompletion of the test results, and in actual commerical use, it isrecommended that some additional cupric⁺⁺ ion or ferric ion be providedin order to assure a complete reaction sequence.

Again, there are many possible and unknown reactions of L-ascorbic acidand toxins, but from a careful review of the observed action accordingto this invention, and from extensive theoretical studies, the aboveeffect is probably at least one of the major reactions taking place inthis invention. In this reaction sequence, ascorbic acid is thereductant, and the cupric⁺⁺ ion is the pro-oxidant which illustrates thereaction. The cupric⁺⁺ ion is reduced to the cuprous⁺ ion (Cu⁺), alongwith molecular oxygen. For each molecule of ascorbic acid that isoxidized to dehydroascorbate, a molecule of hydrogen peroxide isliberated. Hydrogen peroxide is a powerful oxidant when in the presenceof cuprous⁺ ion and is capable of generating hydroxyl radicals accordingto the reaction below:

    Cu.sup.+ -le.sup.- +H.sub.2 O.sub.2 --Cu.sup.++ +OH+HO.sup.-

On the product side of the equation, the hydroxyl free radical ( OH)that is formed is very reactive and is known to participate in reactionsthat irreversibly inactivate proteins. It is recognized that a varietyof metals found in trace quantities in biological systems, as well aswater supplies, may help facilitate the toxin inactivation reactions byascorbic acid. While the cupric ion reactions are illustrated above, nospecial exclusivity should be assigned to the cupric ion, since thistransition series metal should be substituted by a variety of metalsoccurring in trace amounts in biological systems.

Additional Studies with the Ascorbic Acid Antitoxin

The efficacy and specificity of ascorbic acid as an antitoxin has beentested in a rabbit model according to test procedures by John Craig."Serological Evidence for the Identity of Vascular Permeability Factorand Ileial Loop Toxin V. Cholera," Journal of Infectious Diseases, Vol.121, No. 3, 1970, pp. 242,250.) This model has been used extensivelysince its discovery to study the mode of action of bacterial toxins. Ithas also been used to evaluate the means by which toxin activity can beinactivated. This test involves injecting minute amounts of toxin intothe shaved skin of rabbits. The action of toxins is to increase thecapillary permeability of the skin, producing a localized edema. Thisedema is visualized as a wheel of 5-10 mm in diameter. The vascularleakage is made more apparent by injecting a blue dye (i.e. Evans blue)intravenously. As a result, the dye leaks out of the capillaries whosepermeability has been increased by the toxin. The size of the blue spotin the skin is an indicator of the toxin's presence and activity inaltering capillary permeability.

The experimental procedure is essentially that described by Craig. Theborate-gel buffer described can be replaced by phosphate buffered saline(40 mM), at pH 7.0-7.5. Cholera toxin was chosen from a variety oftoxins as the representative toxin for study. Cholera toxin and E. colitoxin are regarded as chemically similar. The amount of cholera toxinfound to elicit the vascular leakage in the skin was 1 to 5 ng/0.1 ml ofthe phosphate buffered saline or borate gel. L-ascorbic acid, when mixedwith the toxin, was effective in blocking the toxins' deleteriousaction. L-ascorbic acid was added in the dose range of 0.001 to 0.1 μMole per 0.1 ml of toxin (1-5 ng). Transition metals, such as CuII andFeIII are thought to be required in trace amounts. Since trace amountsof transition metals are found in biological fluids (i.e. extracellularfluid), the addition of these trace metals was not necessary. Highlevels of metals, i.e. millimolar range, are toxic to biological systemsand should not be used. The specificity of the antitoxin was studied,substituting pyruvic acid and malic acid for the ascorbic acid. Neitherpyruvic acid nor malic acid was effective an antitoxin at even thehighest dose (1 mg/ml) used.

The rabbit skin model has been shown to be predictive of bacterial toxinactivity for many years. It is concluded from the above data thatL-ascorbic acid and its salts are effective and specific antitoxinmaterials. Other similar organic acids, such as pyruvic and malic acids,are not effective as antitoxins.

EXAMPLE C

Using a rabbit model, the rabbits were properly prepared for surgery,then opened to expose the intestines. Several segments of intestineswere created by suturing at spaced intervals. This procedure isolatedthe segments, but otherwise provided a short term normal function.

Into one segment was injected the toxin of E. coli. Into a separatesegment was injected the resultant of a mixture of the same toxinincubated one hour with ascorbic acid.

The rabbits were closed for 24 hours, and when reopened, it was observedthat the segments with toxin were expanded by fluids, but those segmentswith the incubated ascorbic acid were unaffected.

Hence, it is reaffirmed that introduction of ascorbic acid, saltsthereof, or derivatives such as ascorbyl palmetate if introduced to thelower duedenum by any means, will effectively eliminate the otherwisedeleterious effects of toxins provided by various elaborating bacteria.

EXAMPLE D

Further studies of the effects of Vitamin C on cholera enterotoxin havebeen made, using the rabbit skin vascular permeability assay asoriginally described by Dr. John P. Craig (supra). The structure of theE. coli. enterotoxin is very similar to that of cholera enterotoxin, itsmode of action appears to be identical, and its effect on vascularpermeability of rabbit skin is identical.

The study to-date has indicated that when the cholera enterotoxin in adoes of 5 microgram/ml is incubated for one hour with 1 milligram perliter of ascorbic acid prior to intradermal injection in the rabbitskin, the size of the lesion (as determined by the diameter of the lumenarea which develops after intravenous injection of dye, according to thetechnique of Craig) is reduced by 40%. Control areas in the same animal,in which borate-gelatin buffer control was injected into the skin, wereentirely negative both with and without ascorbic acid.

EXAMPLE E

In order to place the ascorbate into position to serve as an effectiveantitoxin, it must be placed in the area where lesions may form at thetime toxins are known to form. Accordingly, it is desirable to place thecrystalline form of the ascorbate in a drug carrier device that mayreside in the intestinal tract in the area colonized by pathogenic E.coli or V. cholerae. Enteric coated time-release dose forms that releasethe ascorbate throughout the length of the small intestine are adequate.The typical dose of ascorbate should range from 15-100 mg/kg bodyweight.

The foregoing theory of the action of ascorbic acid forms will providethe best available guide to those using and prescribing this means ofavoiding the discomfort and danger of toxic diarrhea. Whether the theoryis later confirmed, modified, or overturned, it remains that thisinvention is in the discovery that ascorbic acid, delivered to the lumenof the gut, by any means available prior to invasion of bacteria thatelaborate toxins, will eliminate the toxin and its deleterious effect onthe human system.

This invention directs the placement of ascorbic acid in the lumen ofthe gut whenever one suspects that exposure to toxin elaboratingbacteria may be encountered.

EXAMPLE F

Ascorbic acid formulated into a non-absorbable form remains within thelumen of the gut and remains available to carry out the deactivation ofbacterial toxins. The preferred method is to use ascorbyl palmitate as acomponent in a liposome. The procedure is as follows:

(1) Form the lipid crust

(a) dissolve 20 mg ascorbyl palmitate 60 mg distearoyl lecithin 10 mgcholesterol in 10 ml chloroform: methanol (2:1 v/v).

(b) evaporate solvent to dryness with rotoevaporator at 60° C.

(2) Add 20 ml saline to lipid crust and sonicate at high setting, usinga Heat Systems Sonifier and Branson Cuphorn for 10 minutes at 60° C.

(3) Anneal 10 minutes at 60° C.

The product is a mixture of liposomes (small to large - unilamellar tomultilamellar) that are suitable for oral administration either as aliquid suspension or freeze-dried product in capsules.

A typical dose is 0.2 ml per kg body weight with a range of 0.05 to 1.0ml being acceptable.

The product has reactive ascorbate on the surface as well as within theliposomal layers. The product remains primarily within the lumen of thegut and detoxifies the bacterial toxins without killing organisms.

The available literature does not provide any suggestion of ascorbicacid forms which deliver the ascorbic acid in therapeutic strength tothe lower intestine. There is no known product or literature suggestingneed for such forms of ascorbic acid.

Ascorbic acid in time-release units is available. This time-release formis intended to be taken into the blood stream over an extended timeperiod by absorption from the stomach and duodenum. It is not apparentthat the ascorbic acid need be delivered to the lower intestine, and infact, little would be absorbed from the lower intestine if some of theunits were found intact therein. Therefore, according to the intent oftimerelease of ascorbic acid, any yet intact in the lower intestine iswasted.

This invention provides full dosage of the ascorbic acid into the lowerportion of the duodenum and the intestine. None is intended to beabsorbed in the stomach or upper duodenum.

What is claimed:
 1. The method of eliminating diarrhea caused by thetoxins produced by V.Cholera or E. Coli by the oral administration of anantitoxin effective amount of ascorbic acid or ascorbyl palmitatewhereby ascorbic acid or ascorbyl palmitate is present throughout thelumen of the small and large intestine in a detoxifying amount withoutkilling said organisms.